Method for detecting pathogenic mycobacteria in clinical specimens

Abstract

The present invention relates to detection of pathogenic mycobacteria in clinical specimens such as sputum, cerebrospinal fluid, gastric lavage and tissue biopsies etc., wherein the novel stretch of DNA that lies in the intergenic region between methyl mycolic acid synthase genes mmaA1 and mmaA2 and the flanking region in mmaA1 and mmaA2 genes and is the invention uses a pair of designed oligonucleotide primers that specifically amplifies the target DNA from the clinical specimens.

1. A method for detecting pathogenic mycobacteria in a clinical specimen, said method comprising the steps of:
 (a) clarifying the clinical specimen from contaminant by conventional methods, 
 (b) treating the processed clinical specimen obtained in step (a) with a lysis buffer comprising guanidinium isothiocyanate to inactivate live pathogenic mycobacteria to make the process safe for the user, 
 (c) extracting genomic DNA from the processed clinical specimen obtained from step (b) using a buffer comprising sodium chloride to increase the yield and quality of DNA, 
 (d) designing and synthesizing a set of specific oligonucleotide primers of SEQ ID NO: 5, which is the forward primer and SEQ ID NO: 6, which is the reverse primer, 
 (e) PCR amplifying the genomic DNA from the processed clinical specimen to amplify the DNA stretch having SEQ ID NO: 4 using the specific oligonucleotide primers having SEQ ID NO: 5 and SEQ ID NO: 6, and 
 (f) analyzing the amplified PCR product by restriction fragment length polymorphism (RFLP) analysis for differentiation of the species of the pathogenic mycobacterium for a quick assessment of HIV co-infection. 
 
     
     
       2. A method as claimed in  claim 1 , wherein the clinical specimen is selected from the group consisting of sputum, gastric lavage, cerebrospinal fluid, blood, tissue biopsies, bone marrow aspirates and other body fluids or tissues. 
     
     
       3. A method as claimed in  claim 1 , wherein clarification of the clinical specimen in step (a) from the contaminants is carried out by adding to said specimens a digestion decontamination mix containing mild alkali, NaOH, tri sodium citrate and a mucolytic agent and guanidinium isothiocyanate in the range of about 0.4-2.5 M followed by concentrating the specimens by centrifugation. 
     
     
       4. A method as claimed in  claim 3 , wherein the digestion decontamination mix contains mild alkali, NaOH, tri sodium citrate and a mucolytic agent and guanidinium isothiocyanate is in the range of about 0.5-2.0 M. 
     
     
       5. A method as claimed in  claim 1 , wherein the DNA in step (c) is extracted from the treated clinical specimen using a buffer comprising guanidinium isothiocyanate in a range of about 0.5-8 M, Tris.Cl pH 7.6 in a range of about 20-100 mM, N lauryl Sarcosyl in a range of about 0.5-2% by weight of the buffer, EDTA in a range of about 0.1-20 mM, β-Mercaptoethanol in a range of about 1-25 mM and NaCl is present in an amount of about 0.2M; and purifying the DNA to improve yield by thorough precipitation by organic solvents. 
     
     
       6. A method as claimed in  claim 5 , wherein guanidinium isothiocyanate is about 4M, Tris-HCl pH 7.6 is about 50 mM, N lauryl Sarcosyl is 100 by weight of the buffer, EDTA 1 mM, β-Mercaptoethanol is about 10 mM and NaCl is about 0.2M. 
     
     
       7. A method as claimed in  claim 1 , wherein the genomic DNA yield is increased 25 to 50%. 
     
     
       8. A method as claimed in  claim 1 , wherein amplification of DNA in step (e) is achieved by Touch Down PCR cycling conditions, said conditions comprising steps of providing an initial high annealing temperature in the range of 62-72° C. followed by lowering of temperature in the range of 0.1-1° C. per PCR cycle for the first 10-25 cycles, then subsequently carrying out 30 PCR cycles at an optimum annealing temperature of 56-62° C. 
     
     
       9. A method as claimed in  claim 1 , wherein amplification of DNA in step (e) is achieved by Touch Down PCR cycling conditions, said conditions comprising steps of providing an initial high annealing temperature of 70° C. followed by lowering of temperature of 0.8° C. per PCR cycle for about first 14 cycles to about 58° C. for another 25 PCR cycles. 
     
     
       10. A method as claimed in  claim 1 , wherein the forward primer is SEQ ID NO: 5 and the reverse primer is SEQ ID NO: 6. 
     
     
       11. A method as claimed in  claim 1 , wherein the length of oligomeric primers is between 5 and 100 bases. 
     
     
       12. A method as claimed in  claim 1 , wherein the lysis buffer provides a cleaner preparation of the DNA. 
     
     
       13. A method as claimed in  claim 1 , wherein treatment with the lysis buffer containing 4M guanidinium isothiocyanate inactivates the live mycobacteria to make the procedure safer for the operator. 
     
     
       14. A method as claimed in  claim 1 , wherein the contaminant clarified in step (a) comprises mucus and/or live organisms other than mycobacteria. 
     
     
       15. A set of primers
 consisting of SEQ ID NO: 5 and SEQ ID NO: 6.